The LDSB investigates the organization and activities of developmental regulatory networks using formation of the Drosophila embryonic heart and body wall muscles as a model system. The overarching goal of this work is to comprehensively identify and characterize the upstream regulators of cell fate specification, the downstream effectors of differentiation, and the complex functional interactions that occur among these components during organogenesis. To achieve this objective, we combine contemporary genome-wide experimental and computational approaches with classical genetics and embryology to generate mechanistic hypotheses that we then test at single cell resolution in the intact organism. As a first step toward understanding the structure and function of a developmental regulatory network, the component parts must be identified and assembled into coherent, functionally related groups at the level of single cells. To accomplish this goal, we have undertaken extensive profiling of the genetic programs of various subtypes of mesodermal cells by combining genome-wide transcript profiling with in situ hybridization and antibody-based staining approaches. The integrated strategy that we developed for identifying sets of co-expressed genes in various mesodermal cell types involves the following steps: (i) targeting green fluorescent protein (GFP) to the entire mesoderm or to subsets of mesodermal cells in both wild-type (WT) embryos and embryos from strains in which gain- and loss-of-function genetic manipulations perturb mesoderm development and gene expression patterns in predictable and informative ways; (ii) using flow cytometry to purify GFP-positive and negative cells from strains of each genotype; (iii) qualitative and quantitative profiling of mRNA transcripts using Affymetrix GeneChips; (iv) identifying transcripts that are enriched in the WT mesoderm; (v) pooling the microarray data across genotypes to yield a compendium of expression profiles; (vi) performing a statistical meta-analysis of the combined datasets based on expected trends in gene expression and the relative contribution of each genotype to the detection of appropriate training sets of co-expressed genes; (vii) validating the genome-wide data by whole-embryo in situ hybridization in both WT and selected mutant backgrounds, and (viii) establishing gene co-expression patterns using mutliple RNA and antibody probes to detect the co-localization of transcripts or proteins in the same cells. Collectively, these approaches have enabled us to obtain an accurate spatiotemporal map of gene expression during heart and muscle development, which is a critical first step in studying the molecular mechanisms that are responsible for co-regulating gene expression in individual embryonic cells. Using this research strategy, we initially characterized large sets of genes having restricted expression in somatic muscle founder cells (FCs) and fusion-competnt myoblasts (FCMs). We then added microarray data from purified WT dorsal mesodermal cells, and performed independent meta-analyses for genes expressed in the early cardiac mesoderm (CM). Of 136 randomly selected genes that provided informative in situ hybridization results among the top-ranked 400 candidates and that did not include any of the training set genes, 70 were expressed in the CM and/or in the differentiated heart. Thus, the meta-analysis predicted cardiac genes with an accuracy of 51.4%. Further analyses revealed that 37 genes are expressed in both the CM and the mature heart, 22 genes are expressed in the CM but not in the heart, and 11 genes are expressed in the heart but not in the CM. Numerous genes were also found to be expressed in specific subsets of differentiated heart cells. To gain insight into the biological processes in which these cardiac genes are involved, the entire set was queried for the relative enrichment of Gene Ontology (GO) terms. Overrepresented terms include categories associated with mesoderm development, cardiac differentiation, cell fate specification, transcriptional regulation, cell migration, tube morphogenesis, and the receptor tyrosine kinase (RTK)/Ras pathway, the latter result consistent with prior knowledge of the importance of RTK signaling in heart development. Given independent evidence from another project in which we established the functional importance of Forkhead (Fkh) domain transcription factors (TFs) in cardiogenesis, we overexpressed jumu, a TF belonging to the Fkh family with known heart expression, in the mesoderm. This experiment revealed that a statistically significant fraction of our previously determined set of heart genes are upregulated by increased levels of jumu. Furthermore, a GO analysis demonstrated that jumu-responsive genes are enriched for those involved in asymmetric and symmetric cell division, cell cycle control, and cytokinesis, suggesting one mechanism by which jumu exerts its cardiogenic activity. We also targeted mesodermal overexpression of three FC identity homeodomain (FCI-HD) transcription factors (TFs), Slou, Msh and Eya, the latter being a Six4 co-activator whose ectopic expression serves as a surrogate for activity of this FCI-HD protein. We found that FC gene expression is differentially regulated in a cell-specific manner by HD TFs, and responsiveness correlates with TF co-expression in WT embryos. In addition, HD TFs activate genes uniquely expressed in FCMs, which do not normally contain these TFs, suggesting that FCI-HDs regulate two distinct temporal waves of myogenic gene expression, one in the developing FC and a second in the corresponding mature myotube. Collectively, these results provide an essential and substantive framework for our more recent, in-depth computational and experimental studies of both the structure and function of myogenic and cardiogenic transcriptional regulatory networks.